It is well known that ultrasonic imaging comprises a valuable diagnostic tool, for example in studies of the vascular system, particularly in cardiography, and of tissue microvasculature. A variety of ultrasound contrast media has been proposed to enhance the acoustic images so obtained, including suspensions of solid particles, emulsified liquid droplets, gas bubbles and encapsulated gases or liquids. The most successful ultrasound contrast media have generally consisted of dispersions of small bubbles of gas that can be injected intravenously. For example WO 97/29783 and WO93/05819 describe such microbubble dispersions. If appropriately stabilised, microbubbles may permit highly effective ultrasound visualisation of, for example, the vascular system and tissue microvasculature, often at advantageously low doses. Such contrast media typically include a material stabilising the gas, for example emulsifiers, oils, thickeners or sugars, or by entraining or encapsulating the gas in a variety of systems, e.g. as porous gas-containing microparticles or as encapsulated gas microbubbles. The microbubbles include a gas that is essential for the performance of the ultrasound contrast agent, and a variety of gases have been found to enhance properties such as the microbubble stability and duration of echogenic effect. One group of ultrasound contrast media is prepared and delivered as a ready-made preparation comprising a liquid composition of encapsulated gas microbubbles.
Various processes can be used to prepare microbubbles. Such gas-containing microbubbles may be produced by shaking or sonicating a liquid containing a membrane-forming material in the presence of a suitable gas or gas mixture. Other processes include spray drying. However, the microbubbles produced by such techniques have a broad size distribution which may vary from batch to batch and moreover the yield, i.e. the percentage of membrane forming material which ends up in appropriately sized microbubbles, may also vary from batch to batch. U.S. Pat. No. 5,552,133 describes a process for making encapsulated gas microspheres comprising a heat-denaturable protein encapsulating a gas, using a colloid mill. An aqueous solution of a heat-denaturable protein is combined with a gas, and is mixed by applying mechanical shear forces to the mixture to form a suspension of gas microbubbles, wherein the protein becomes denatured and deposited at the gas-solution interface.
When preparing microbubbles it is important to have a robust process that repeatedly provides a product according to product specifications. Desirably the microbubbles produced will have a narrow size distribution about the desired microbubble size, generally 1 to 7 μm, e.g. 3-5 μm. Desirable, the percentage of large microbubbles, such as above 7 μm should be minimal and well limited. To achieve this, the standard deviation of the microbubble sizes should be small. This has not been achieved when using the processes of the prior art. One challenge is to produce reproducible microbubbles having a narrow size distribution throughout the process cycle and each time the process is run. A robust process for preparing contrast media, such as for preparing a composition comprising gas microbubbles encapsulated by a denatured protein, has been sought.